Coding Sequence Mutations in the Alpha Subunit of Propionyl-CoA Carboxylase in Patients with Propionic Acidemia

Abstract

Propionic acidemia is a rare autosomal recessive disorder of intermediary metabolism. It is caused by a deficiency of the mitochondrial enzyme propionyl-CoA carboxylase (PCC, EC 6.4.1.3), a heteropolymeric protein composed of two subunits, α and β. PCC requires ATP and biotin as cofactors for the reaction, the latter enzymatically added onto the α subunit. We investigated coding sequence mutations in the α subunit of PCC by analyzing fibroblast RNA from propionic acidemia patients deficient in α subunit function by single-strand conformation polymorphism and direct sequencing. Five missense mutations and one short in-frame deletion were found among different patients. Four mutations were located in the putative biotin carboxylase domain, whereas the two others were within the 67-amino-acid C-terminal domain previously shown to be required to obtain biotinylation of the α subunit. We analyzed fibroblast extracts for the presence of a biotinylated α subunit by Western blot analysis using streptavidin coupled to alkaline phosphatase. Four of five cell lines failed to show a biotinylated α subunit, regardless of the position of the mutations within the coding sequence. Two mutations located in the biotinylation domain were expressed in anEscherichia coli-based system and shown to abolish biotinylation of the domain. The results suggest that most mutations have a severe impact on the stability or the functionality of the α subunit.